Many lines of evidence have shown that modification of cytosine bases residing in the dinucleotide sequence CpG in vertebrate genomes plays an essential role in regulating a variety of genome functions such as X chromosome inactivation, parental imprinting, inactivation of genomic retroelements, and differential gene expression. Across the human genome, about 80% of the CpG dinucleotides are heavily methylated, but some areas remain unmethylated, preferentially in the GC rich CpG islands [Bird, A. P., CpG-rich islands and the function of DNA methylation. Nature, 1986. 321(6067): p. 209-13.]. DNA methylation can perform its regulatory function through the differential marking of genes. Cytosine methylation is a stable but potentially reversible process that allows for the temporal and spatial-specific regulation of gene in higher organisms.
Several different strategies have been applied to detect methylated CpG dinucleotides in eukaryotic genomes (reviewed in [van Steensel, B. and S. Henikoff, Epigenomic profiling using microarrays. Biotechniques, 2003. 35(2): p. 346-50, 352-4, 356-7]). The most frequently used method is the bisulfite modification-based strategy, developed by Frommer et al. [ Frommer, M., et al., A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci USA, 1992. 89(5): p. 1827-31.]. In this method, bisulfite converts unmethylated cytosine bases to uracil, whereas methylated cytosines remain unaltered. Such sequences can be directly sequenced using the Sanger sequencing method or can be interrogated using microarrays. In such microarrays, oligonucleotide pairs that differ by having either a cytosine or a thymine at a methylatable position of a cytosine can discriminate the two nucleotides by incubating at a temperature that allows only exact matches between the probe and the oligonucleotide Adorjan, P., et al., Tumour class prediction and discovery by microarray-based DNA methylation analysis. Nucleic Acids Res, 2002. 30(5): p. e21; Gitan, R. S., et al., Methylation-specific oligonucleotide microarray: a new potential for high-throughput methylation analysis. Genome Res, 2002. 12(1): p. 158-64; Balog, R. P., et al., Parallel assessment of CpG methylation by two-color hybridization with oligonucleotide arrays. Anal Biochem, 2002. 309(2): p. 301-10; Hou, P., et al., A microarray to analyze methylation patterns of p16(Ink4a) gene 5′-CpG islands. Clin Biochem, 2003. 36(3): p. 197-202.
Several other methods of providing methylation status on a global scale including microarray experiments have been published. In a method called differential methylation hybridization (DMH) [Huang, T. H., U.S. Pat. No. 6,605,432 B1 issued Aug. 12, 2003.], genomic DNA (gDNA) from breast cancer cells were treated with the four-base cutter MseI that restricts gDNA into small fragments of 100-200 bp. This enzyme rarely cuts in CpG-rich regions, leaving many CpG islands intact. MseI cleavage is followed by ligation of end adaptors specific for MseI sticky-ends, cleavage with the methylation-sensitive enzyme BstUI, and subsequent PCR amplification. This method results in amplification of the hypermethylated fraction of gDNA, and ignores the hypomethylated or unmethylated fraction.
Microarrays in this study contains DNA fragments representing various CpG islands. Several other publications used the step of enrichment for the hypermethylated fraction of a given genome [Yan, P. S., et al., Applications of CpG island microarrays for high-throughput analysis of DNA methylation. J Nutr, 2002. 132(8 Suppl): p. 2430S-2434S;. Yan, P. S., et al., Use of CpG island microarrays to identify colorectal tumors with a high degree of concurrent methylation. Methods, 2002. 27(2): p. 162-9; Shi, H., et al., Triple analysis of the cancer epigenome: an integrated microarray system for assessing gene expression, DNA methylation, and histone acetylation. Cancer Res, 2003. 63(9): p. 2164-71; Toyota, M., et al., Identification of differentially methylated sequences in colorectal cancer by methylated CpG island amplification. Cancer Res, 1999. 59(10): p. 2307-12; Yan, P. S., et al., Dissecting complex epigenetic alterations in breast cancer using CpG island microarrays. Cancer Res, 2001. 61(23): p. 8375-80]. Amplification of non-methylated sequences is suppressed by the digestion of the template DNA before PCR with the restriction enzymes BstUI and HpaII, which are blocked by methylation of their target sequence [Yan, P. S., et al., Dissecting complex epigenetic alterations in breast cancer using CpG island microarrays. Cancer Res, 2001. 61(23): p. 8375-80.]. The resulting hypermethylated DNA fraction was used to compare the methylation patterns from tumor and control tissues by hybridizing to microarrays containing randomly cloned genomic fragments that were enriched in CpG islands
A related method uses a digestion step with SmaI, followed by digestion with XmaI, which is a methyl-insensitive isoschizomer of SmaU [Hatada, I., et al., A Microarray-based method for detecting methylated loci. J Hum Genet, 2002. 47(8): p. 448-51.]. The cleavage with SmaI produces blunt end DNA fragment, whereas the cleavage products of XmaI contains protruding ends, which are ligated to specific XmaI-adaptors. After a PCR that uses primers specific for these adaptors, the resulting amplification products, which consist mainly of methylated 5′-CCCGGG-3′ sequences, are hybridized to microarrays.
Another method that uses methylation-sensitive restriction enzymes for fractionating DNA was presented by Tompa et al. [Tompa, R., et al., Genome-wide profiling of DNA methylation reveals transposon targets of CHROMOMETHYLASE3. Curr Biol, 2002. 12(1): p. 65-8.]. This strategy used the methylation sensitive enzyme MspI, which cleaves 5′CCGG-3′ but is blocked by methylation of the outer cytosine (m5′-CCGG-3′). Digested DNA samples were size-fractionated on sucrose gradients (5%-30%) by ultracentrifugation as previously described [van Steensel, B., J. Delrow, and S. Henikoff, Chromatin profiling using targeted DNA adenine methyltransferase. Nat Genet, 2001. 27(3): p. 304-8.]. Gradient fraction containing plant DNA fragments smaller than 2.5 kb, as determined by gel-electrophoresis, were pooled and concentrated by isopropanol precipitation. Tester and control samples were then labeled with Cy3- or Cy5-dCTP by random priming and co-hybridized to microarrays that contained spotted PCR amplification products that primarily represented randomly chosen locations from the Arabidopsis genome [Tompa, R., et al., Genome-wide profiling of DNA methylation reveals transposon targets of CHROMOMETHYLASE3. Curr Biol, 2002. 12(1): p. 65-8].
Wang (WO 03/027259, published Apr. 3, 2003) discloses cleavage of mouse genomic DNA with the methyl-sensitive enzyme HpaII, ligation of adaptors specific for HpaII sticky-ends, and PCR using Cy-3 or Cy-5 labeled primers. The amplicons produced by this method may retain methylated sequences that are in between the cleaved HpaII restriction sites.
Martienssen et al. (US 2004/0132048, published Jul. 8, 2004) suggest methods to obtain methylated or unmethylated fractions of genomic DNA obtained by cleavage with methyl-dependent enzymes such as McrBC that specifically cleave methylated sequences, or by cleavage with, for example, HpaII which does not cleave methylated sequences. Similar to Wang, the Martienssen et al. methods may be complicated by retention of methylated sequences between unmethylated restriction sites. Furthermore, there may be retention of unmethylated sequences between the methylated restriction sites. Another drawback of these methods is a step of physically separating the cleaved methylated or unmethylated fractions from the rest of the genomic DNA by gel electrophoresis, size exclusion chromatography and size differential centrifugation in a sucrose gradient. Methods using a physical separation step require relatively large amounts of starting material due to inefficiencies of DNA recovery inherent in the separation step.
There is a need in the art to develop new methods and systems for epigenetic profiling. Further there is a need in the art for new methods and systems for epigenetic profiling of chromosomes and genomes. Further still, there is a need in the art to develop methods and systems to assess methylation levels of probed loci such as repetitive elements, genes, imprinting elements, promoters, enhancer elements, intron sequences and whole genomes.